The invention relates to block copolymer compositions and methods for intramuscular administration of polynucleotides.
The unique features of smooth, skeletal, and cardiac muscles, have presented numerous challenges for the development and administration of effective polynucleotide compositions for intramuscular administration. Direct injection of purified plasmids (xe2x80x9cnaked DNAxe2x80x9d) in isotonic saline into muscle was found to result in DNA uptake and gene expression in smooth, skeletal, and cardiac muscles of various species. Rolland A., Critical Reviews in Therapeutic Drug Carrier Systems, Begell House, 143 (1998). It is believed that the unique cytoarchitectural features of muscle tissue are responsible for the uptake of polynucleotides because skeletal and cardiac muscle cells appear to be better suited to take-up and express injected foreign DNA vectors relative to other types of tissues. Dowty and Wolff, Gene Therapeutics: Methods and Applications of Direct Gene Transfer, Birkhxc3xa4user, Boston, p.182 (1994). The relatively low expression levels attained by this method, however, have limited its applications. See Aihara and Miyazaki, Nature Biotechnology, 16:867 (1998). Additionally, traditional gene delivery systems such as polycations, cationic liposomes, and lipids that are commonly proposed to boost gene expression in other tissues usually result in inhibition of gene expression in skeletal and cardiac muscles. Dowty and Wolff, Gene Therapeutics: Methods and Applications of Direct Gene Transfer, Birkhxc3xa4user, Boston, p. 82 (1994).
Anionic polymers such as dextran sulfate and salmon DNA can decrease gene expression in the muscle. Rolland A., Critical Reviews in Therapeutic Drug Carrier Systems, Begell House, 1998, p. 143. Various noncondensive interactive polymers have been used with a limited success to modify gene expression in striated muscle. Nonionic polymers such as poly(vinyl pyrrolidone) poly(vinyl alcohol) interact with plasmids through hydrogen bonding. Rolland A., Critical Reviews in Therapeutic Drug Carrier Systems, Begell House, 1998, p. 143. These polymers may facilitate the uptake of polynucleotides in muscle cells and cause up to 10-fold enhancement of gene expression. However, to achieve a significant increase in gene expression, high concentrations of polymers (about 5% and more) need to be administered. Mumper et al., Pharmacol. Res., 13, 701-709 (1996); March et al., Human Gene Therapy, 6(1), 41-53 (1995). This high concentration of poly(vinyl pyrrolidone) poly(vinyl alcohol) needed to improve gene expression can be associated with toxicity, inflammation, and other adverse effects in muscle tissues. Block copolymers have been used to improve gene expression in muscle or to modify the physiology of the muscle for subsequent therapeutic applications. See U.S. Pat. Nos. 5,552,309; 5,470,568; 5,605,687; and 5,824,322. For example, block copolymers can be used in a gel-like form (more than 1% of block copolymers) to formulate virus particles used to perform gene transfer in the vasculature. In the same range of block copolymers concentration (1-10%), it is possible with block copolymer to modify the permeability of damaged muscle tissue (radiation and electrical injury, and frost bite). In addition DNA molecules can be incorporated into cells following membrane permeabilization with block copolymers. For these applications, block copolymers were used at concentrations giving gel-like structures and viscous delivery systems. These systems are unlikely to enable diffusion of the DNA injected into the muscle, however, thus limiting infusion of the DNA into the myofibers.
There is thus a need for compositions and methods increasing efficacy of polynucleotides expression upon administration in the muscle.
Beside the need to improve gene expression in muscle other tissues in the body would benefit from a gene transfer in a situation when there is a genetic disorder, and/or an abnormal over-expression of a gene, and/or absence of a normal gene. Several polynucleotides such as RNA, DNA, viruses, ribozymes can be used for gene therapy purposes. However, many problems, like the ones described below, have been encountered for successful gene therapies.
The use of antisense polynucleotides to treat genetic diseases, cell mutations (including cancer causing or enhancing mutations) and viral infections has gained widespread attention. This treatment tool is believed to operate, in one aspect, by binding to xe2x80x9csensexe2x80x9d strands of mRNA encoding a protein believed to be involved in causing the disease site sought to be treated, thereby stopping or inhibiting the translation of the mRNA into the unwanted protein. In another aspect, genomic DNA is targeted for binding by the antisense polynucleotide (forming a triple helix), for instance, to inhibit transcription. See Helene, Anti-Cancer Drug Design, 6:569 (1991). Once the sequence of the mRNA sought to be bound is known, an antisense molecule can be designed that binds the sense strand by the Watson-Crick base-pairing rules, forming a duplex structure analogous to the DNA double helix. Gene Regulation: Biology of Antisense RNA and DNA, Erikson and lxzant, eds., Raven Press, New York, 1991; Helene, Anti-Cancer Drug Design, 6:569 (1991); Crooke, Anti-Cancer Drug Design, 6:609 (1991). A serious barrier to fully exploiting this technology is the problem of efficiently introducing into cells a sufficient number of antisense molecules to effectively interfere with the translation of the targeted mRNA or the function of DNA.
The invention relates to compositions of polynucleotides, such as RNA, DNA or their derivatives, and block copolymers. These compositions are useful for gene therapy purposes, including gene replacement or excision therapy, and gene addition therapy, vaccination, as well as therapeutic situations in which it is desirable to express or down-regulat a polypeptide in the body or in vitro.
Preferred embodiments include compositions having polynucleotides and block copolymers with cationic segments as well as compositions having polynucleotides and nonionic polyether block copolymers. In one embodiment, particularly useful for intramuscular administration, polynucleotides are formulated with block copolymers of poly(oxyethylene) and poly(oxypropylene).
The compositions of the current invention provide an efficient vehicle for introducing polynucleotides into a cell, protecting polynucleotides against degradation in body fluids, transport of polynucleotides across biological membranes and biological barriers (such as the blood-brain barrier, blood-cerebral fluid barrier, and intestinal barrier), modification of biodistribution of polynucleotides in the body and enhancement of gene expression including selective gene expression in various tissues and organs in the body of human or animal.
The invention further relates to methods of inserting polynucleotides into cells utilizing the compositions of the invention, and methods of treatment having administering these compositions in humans and animals.
In a preferred embodiment, the block copolymer conforms to one of the following formulae: 
wherein A and Axe2x80x2 are A-type linear polymeric segments, B and Bxe2x80x2 are B-type linear polymeric segments, and R1, R2, R3, and R4 are either block copolymers of formulas (I), (II), or (III), or hydrogen and L is a linking group, with the proviso that no more than two of R1, R2, R3, or R4 are hydrogen.
In another preferred embodiment, the block copolymers are poly(oxyethylene) and poly(oxypropylene) chain segments. In yet another preferred embodiment, the polynucleotide compositions have polycationic polymers having a plurality of cationic repeating units. In this case, the polynucleotides can be complexed with the polycation and stabilized in the complex. These compositions demonstrate increased permeability across cell membranes and are well suited for use as vehicles for delivering nucleic acid into cells.
In another embodiment, the invention relates to polynucleotide compositions having:
(a) a polynucleotide or derivative thereof;
(b) a block copolymer having a polyether segment and a polycation segment, wherein the polyether segment comprises at least an A-type block, and the polycation segment comprises a plurality of cationic repeating units.
In a preferred second embodiment, the copolymer relates to polymers of formulae: 
wherein A, Axe2x80x2, and B are as described above, wherein R and Rxe2x80x2 are polymeric segments having a plurality of cationic repeating units, and each cationic repeating unit in a segment is the same or different from another unit in the segment. The polymers of this embodiment can be termed xe2x80x9cpolynonion/polycationxe2x80x9d polymers. The R and Rxe2x80x2, blocks can be termed xe2x80x9cR-typexe2x80x9d polymeric segments or blocks. The polynucleotide compositions of this embodiment provide an efficient vehicle for introducing polynucleotides into a cell.
Accordingly, the invention thus further relates to methods of inserting polynucleotide into cells utilizing the compositions of the invention.
In yet another embodiment, the invention relates to polynucleotide compositions having a polynucleotide derivative comprising a polynucleotide segment and a polyether segment attached to one or both of the polynucleotide 5xe2x80x2 and 3xe2x80x2 ends, wherein the polyether comprises an A-type polyether segment.
In a preferred third embodiment, the derivative comprises a block copolymer of formulas: 
wherein pN represents a polynucleotide having 5xe2x80x2 to 3xe2x80x2 orientation, and A, Axe2x80x2, and B are polyether segments as described above. In another preferred third embodiment, the polynucleotide complex comprises a polycationic polymer. The polynucleotide component (pN) of formulas (IX) through (XIII) will preferably have from about 5 to about 1,000,000 bases, more preferably about 5 to about 100,000 bases, yet more preferably about 10 to about 10,000 bases.
The polynucleotide compositions provide an efficient vehicle for introducing polynucleotides into a cell. Accordingly, the invention also relates to methods of inserting polynucleotide into cells the compositions of the invention. In another preferred embodiment, polynucleotides are covalently linked to block copolymers of poly(oxyethylene) and poly(oxypropylene).
Another embodiment of the invention relates to a polyetherpolycation copolymers having a polymer, a polyether segment, and a polycationic segment having a plurality of cationic repeating units of formula xe2x80x94NHxe2x80x94R0, wherein R0 is a straight chain aliphatic group of 2 to 6 carbon atoms, which may be substituted, wherein said polyether segments comprise at least one of an A-type of B-type segment. In another preferred embodiment, the polycation polymer has a polymer according to the following formulae: 
wherein A, Axe2x80x2, and B are as described above, wherein R and Rxe2x80x2 are polymeric segments having a plurality of cationic repeating units of formula xe2x80x94NHxe2x80x94R0xe2x80x94, wherein R0 is a straight chain aliphatic group having from 2 to 6 carbon atoms, which may be substituted. Each xe2x80x94NHxe2x80x94R0xe2x80x94 repeating unit in an R-type segment can be the same or different from another xe2x80x94NHxe2x80x94R0xe2x80x94 repeating unit in the segment.
In yet another embodiment, the invention provides a polycationic polymer having a plurality of repeating units of formula: 
where R8 is:
(1) xe2x80x94(CH2)nxe2x80x94CH(R13)xe2x80x94, wherein n is an integer from 0 to about 5, and R13 is hydrogen, cycloalkyl having 3-8 carbon atoms, alkyl having 1-6 carbon atoms, or (CH2)mR14, where m is an integer from 0 to about 12 and R14 is a lipophilic substituent of 6 to 20 carbon atoms;
(2) a carbocyclic group having 3-8 ring carbon atoms, wherein the group can be for example, cycloalkyl or aromatic groups, and which can include alkyl having 1-6 carbon atoms, alkoxy having 1-6 carbon atoms, alkylamino having 1-6 carbon atoms, dialkylamino wherein each alkyl independently has 1-6 carbon atoms, amino, sulfonyl, hydroxy, carboxy, fluoro, or chloro substituents; or (3) a heterocyclic group, having 3-8 ring atoms, including heterocycloalkyl or heteroaromatic groups from 1 to 4 heteroatoms selected from the group consisting of oxygen, nitrogen, sulfur and mixtures thereto, and which further can include alkyl having 1-6 carbon atoms, alkoxy having 1-6 carbon atoms, alkylamino having 1-6 carbon atoms, dialkylamino wherein each alkyl independently has 1-6 carbon atoms, amino, sulfonyl, hydroxy, carboxy, fluoro or chloro substituents.
R9 is a straight chain aliphatic group of 1 to 12 carbon atoms, and R10, R11, and R12 are independently hydrogen, an alkyl group of 1-4 carbon atoms. R9 preferably is 2-10 carbon atoms, more preferably, 3-8 carbon atoms. R14 preferably includes an intercalating group, which is preferably an acrydine or ethydium bromide group. The number of repeating units in the polymer is preferably between about 3 and 50, more preferably between about 5 and 20. This polymer structure can be incorporated into other embodiments of the invention as an R-type segment or polycationic polymer. The ends of this polymer can further be modified with a lipid substituent.
The monomers that are used to synthesize polymers of this embodiment are suitable for use as the monomers fed to a DNA synthesizer, as described below. Thus, the polymer can be synthesized very specifically. Further, the additional incorporation of polynucleotide sequences, polyether blocks, and lipophilic substituents can be done using the advanced automation developed for polynucleotide syntheses. This embodiment also encompasses the method of synthesizing a polycationic polymer.
In yet another embodiment, the invention relates to polymers having a plurality of covalently bound polymer segments wherein the segments have (a) at least one polycation segment which segment is a cationic homopolymer, copolymer, or block copolymer having comprising at least three aminoalkylene monomers, said monomers being selected from the group consisting of:
(i) at least one tertiary amino monomer of the formula: 
and the quaternary salts of said tertiary amino monomer, and (ii) at least one secondary amino monomer of the formula: 
and the acid addition and quaternary salts of said secondary amino monomer, in which:
R1 is hydrogen, alkyl of 2 to 8 carbon atoms, an A monomer, or a B monomer; each of R2 and R3, taken independently of the other, is the same or different straight or branched chain alkanediyl group of the formula:
xe2x80x94(CzH2z)xe2x80x94
in which z has a value of from 2 to 8; R4 is hydrogen satisfying one bond of the depicted geminally bonded carbon atom; and R5 is hydrogen, alkyl of 2 to 8 carbon atoms, an A monomer, or a B monomer; R6 is hydrogen, alkyl of 2 to 8 carbon atoms, an A monomer, or a B monomer; R7 is a straight or branched chain alkanediyl group of the formula:
xe2x80x94(CzH2z)xe2x80x94
in which z has a value of from 2 to 8; and R8 is hydrogen, alkyl of 2 to 8 carbon atoms, an A monomer, or a B monomer; and
(b) at least one straight or branched chained polyether segment having from about 5 to about 400 monomeric units which is:
(i) a homopolymer of a first alkyleneoxy monomer xe2x80x94OCnH2nxe2x80x94 or
(ii) a copolymer or block copolymer of said first alkyleneoxy monomer and a second different alkyleneoxy monomer xe2x80x94OCmH2mxe2x80x94, in which n has a value of 2 or 3 and m has a value of from 2 to 4.
Polymers of formulas (I), (III), (III), or (IV) can also be mixed with each other or can be mixed either additionally or alternatively with one or more of the polymers of formula (V-a or b), (VI-a or b), (VII-a or b), and (VIII-a or b) and/or with polynucleotide derivatives of formulas (IX-a,b,c, or d), (X-a,b,c,d,e, or f), (XI), (XII) or (XIII) to provide an efficient vehicle for delivering polynucleotide to the interior of cells.
The degree of polymerization of the hydrophilic (A-type) blocks or the hydrophobic (B-type) blocks of formulas (I)-(XIII) can preferably be between about 5 and about 400. More preferably, the degree of polymerization shall be between about 5 and about 200, still more preferably, between about 5 and about 80. The degree of polymerization of the R-type polycation blocks can preferably be between about 2 and about 300. More preferably, the degree of polymerization shall be between about 5 and about 180, still more preferably, between about 5 and about 60. The degree of polymerization of the polycationic polymer can preferably be between about 10 and about 10,000. More preferably, the degree of polymerization shall be between about 10 and about 1,000, still more preferably, between about 10 and about 100.
The repeating units that comprise the blocks, for A-type, B-type and R-type blocks, will generally have molecular weight between about 30 and about 500, preferably between about 30 and about 100, still more preferably between about 30 and about 60. Generally, in each of the A-type or B-type blocks, at least about 80% of the linkages between repeating units will be ether linkages, preferably, at least about 90% will be ether linkages, more preferably, at least about 95% will be ether linkages. Ether linkages, for the purposes of this application, encompass glycosidic linkages (i.e., sugar linkages). However, in one aspect, simple ether linkages are preferred.
In yet another preferred embodiment, the compositions of the invention are useful for gene therapy purposes, including gene replacement or excision therapy, and gene addition therapy, vaccination, and any therapeutic situation in which a polypeptide should be expressed or down-regulated in the body or in vitro. In one aspect of this invention the polynucleotide compositions are used for gene therapy in muscle tissue, including but not limited to smooth, skeletal and cardiac muscles of the human or animals. It is preferred that compositions for intramuscular administration comprise the block copolymers of poly(oxyethylene) and poly(oxypropylene).
In still another preferred embodiment, the invention relates to compositions having at least one poly(oxyethylene) and poly(oxypropylene) block copolymer with oxyethylene content of 50% or less, and at least one poly(oxyethylene) and poly(oxypropylene) block copolymer with oxyethylene content of 50% or more, and a polynucleotide. The preferrable ratio by weight of the block copolymer with oxyethylene content of 50% or less to the block copolymer with oxyethylene content of 50% or more is 1:2, more preferrably 1:5.
It is preferred that the compositions of this invention do not form gels. The dispersions include suspensions, emulsions, microemulsions, micelles, polymer complexes, and real polymers solutions are particularly preferred. In one aspect the concentration of the polymers and block copolymers in the polynucleotide compositions is less that 10%, preferably less that 1%, more preferred less than 0.5%, yet more preferred less than 0.1%.
Block copolymers are most simply defined as conjugates of at least two different polymer segments (Tirrel, M., Interactions of Surfactants with Polymers and Proteins, Goddard E. D. and Ananthapadmanabhan, K. P. (eds.), CRC Press, Boca Raton, Ann Arbor, London, pp. 59-122, (1992). Some block copolymer architectures are below. 
The simplest block copolymer architecture contains two segments joined at their termini to give an A-B type diblock. Consequent conjugation of more than two segments by their termini yields an A-B-A type triblock, A-B-A-B-type multiblock, or even multisegment A-B-C-architectures. If a main chain in the block copolymer can be defined in which one or several repeating units are linked to different polymer segments, then the copolymer has a graft architecture of, e.g., an A(B)n type. More complex architectures include for example (AB)n or AnBm starblocks which have more than two polymer segments linked to a single center.
Formulas XVIII-XXIII of the invention are diblocks and triblocks. At the same time, conjugation of polycation segments to the ends of polyether of formula XVII yields starblocks (e.g., (ABC)4 type). In addition, the polyspermine of examples 13-15 (below) are branched. Modification of such a polycation with poly(ethylene oxide) yields a mixture of grafted block copolymers and starblocks. In accordance with the present invention, all of these architectures can be useful for polynucleotide delivery.
The entire disclosure of U.S. Ser. No. 08/342,079, filed, Nov. 18, 1994, now U.S. Pat. No. 5,783,178 is hereby incorporated herein by reference.
In another aspect, the invention provides a polynucleotide complex between a polynucleotide and polyether block copolymers. Preferably, the polynucleotide complex will further include a polycationic polymer. The compositions can further include suitable targeting molecules and surfactants. In another aspect, the invention provides a polynucleotide complex between a polynucleotide and a block copolymer comprising a polyether block and a polycation block. In yet another aspect, the invention provides polynucleotides that have been covalently modified at their 5xe2x80x2 or 3xe2x80x2 end to attach a polyether polymer segment.
Polycations. Preferred polycation polymers and polycation segments of the copolymers include but are not limited to polyamines (e.g., spermine, polyspermine, polyethyleneimine, polypropyleneimine, polybutilene-imine, poolypentyleneimine, polyhexyleneimine and copolymers thereof), copolymers of tertiary amines and secondary amines, partially or completely quaternized amines, polyvinyl pyridine, and the quaternary ammonium salts of these polycation segments. These preferred polycation fragments also include aliphatic, heterocyclic or aromatic ionenes (Rembaum et al., Polymer letters, 6:159 (1968); Tsutsui, T., Development in ionic polymers-2, Wilson A. D. and Prosser, H. J. (eds.) Applied Science Publishers, London, New York, vol. 2, pp. 167-187, 1986).
The polycationic polymers and the R-type blocks have several positively ionizable groups and a net positive charge at physiologic pH. The polyether/polycation polymers of formulas (V)-(VIII) can also serve as polycationic polymers. Preferably, the polycation segments have at least about 3 positive charges at physiologic pH, more preferably, at least about 6, still more preferably, at least about 12. Also preferred are polymers or segments that, at physiologic pH, can present positive charges with a distance between the charges of about 2 xc3x85 to about 10 xc3x85. The distances established by ethtyleneimine, aminopropylene, aminobutilene, aminopentylene and aminohehhylene repeating units, or by mixtures of at least two of these groups are most preferred. Preferred are polycationic segments that utilize (NCH2CH2), (NCH2CH2CH2), (NCH2CH2CH2CH2), (NCH2CH2CH2CH2CH2), and (NCH2CH2CH2CH2CH2CH2) repeating units, or a mixture thereof.
Polycation segments having an xe2x80x94Nxe2x80x94R0xe2x80x94 repeating unit are also preferred. R0 is preferably an ethylene, propylene, butylene, pentylene, or hexylene which can be modified. In a preferred embodiment, in at least one of the repeating units R0 includes a DNA intercalating group such as an ethidium bromide group. Such intercalating groups can increase the affinity of the polymer for nucleic acid. Preferred substitutions on R0 include alkyl of 1-6 carbon atoms, hydroxy, hydroxyalkyl, wherein the alkyl has 1-6 carbon atoms, alkoxy having 1-6 carbon atoms, an alkyl carbonyl group having 2-7 carbon atoms, alkoxycarbonyl wherein the alkoxy has 1-6 carbon atoms, alkoxycarbonylalkyl wherein the alkoxy and alkyl each independently has 1-6 carbon atoms, alkylcarboxyalkyl wherein each alkyl group has 1-6 carbon atoms, aminoalkyl wherein the alkyl group has 1-6 carbon atoms, alkylamino or dialkylamino where each alkyl group independently has 1-6 carbon atoms, mono- or di-alkylaminoalkyl wherein each alkyl independently has 1-6 carbon atoms, chloro, or chloroalkyl wherein the alkyl has from 1-6 carbon atoms, fluoro, or fluoroalkyl wherein the alkyl has from 1-6 carbon atoms, cyano, or cyano alkyl wherein the alkyl has from 1-6 carbon atoms or a carboxyl group. More preferably, R0 is ethylene, propylene, or butylene.
The polycation polymers and polycation segments in the copolymers of the invention can be branched. For example, polyspermine-based copolymers are branched. The cationic segment of these copolymers was synthesized by condensation of 1,4-dibromobutane and N-(3-aminopropyl)-1,3-propanediamine. This reaction yields highly branched polymer products with primary, secondary, and tertiary amines.
An example of branched polycations are products of the condensation reactions between polyamines containing at least 2 nitrogen atoms and alkyl halides containing at least 2 halide atoms (including bromide or chloride). In particular, the branched polycations are produced as a result of polycondensation. An example of this reaction is the reaction between N-(3-aminiopropyl)-1,3-propanediamine and 1,4-dibromobutane, producing polyspermine.
Another example of a branched polycation is polyethyleneimine represented by the formula:
(NHCH2CH2)x[N(CH2CH2)CH2CH2]y
Additionally, cationic dendrimers, for example, polyamidoamines (Tomalia el al., Angew. Chem., Int. Ed. Engl., 1990, 29, 138) can be also used as polycation segments of block copolymers for gene delivery.
Examples of useful polymers pursuant to formulas (V)-(VIII) include the poly(oxyethylene)-poly-L-lysine) diblock copolymer of the following formula: 
wherein i is an integer of from about 5 to about 100, and j is an integer from about 4 to about 100.
A second example is the poly(oxyethylene)-poly-(L-alanine-L-lysine) diblock copolymer of formula: 
wherein i is an integer of from about 5 to about 100, and j is an integer from about 4 about 100.
A third example is the poly(oxyethylene)-poly(propyleneimine/butyleneimine) diblock copolymer of the following formula: 
wherein i is an integer from about 5 about 200 and j is an integer from 1 to about 10. A fourth example is the poly(oxyethylene)-poly(N-ethyl-4-vinylpyridinium bromide) (xe2x80x9cpOE-pEVP-Brxe2x80x9d) of formula: 
wherein i is an integer of from about 5 to about 100 and j is an integer of from about 10 to about 500. Still another example is the polymer of formula:
CH3Oxe2x80x94(CH2CH2O)iCO[(NH(CH2)3)2NH(CH2)4]jxe2x80x94(NH(CH2)3)2xe2x80x94NHCOxe2x80x94Oxe2x80x94(CH2CH2O)kxe2x80x94CH3xe2x80x83xe2x80x83(XXII)
wherein i is an integer from about 10 to about 200, j is an integer from about 1 to about 8, and k is an integer from about 10 to about 200. Still another example is the polymer of formula:
Hxe2x80x94Gjxe2x80x94(NH(CH2)3)2xe2x80x94Nxe2x80x94NHxe2x80x94COxe2x80x94Oxe2x80x94(CH2CH2O)iCOxe2x80x94Gmxe2x80x94(NH(CH2)3)2xe2x80x94NH2xe2x80x83xe2x80x83(XXIII)
wherein xe2x80x9cGxe2x80x9d comprises xe2x80x94(NH(CH2)3)3xe2x80x94CH2NH2xe2x80x94, i and j are as defined for formula (XVIII), and m is an integer from about 1 to about 8.
Nonionic polyether block copolymers and nonionic polyether segments. Nonionic polyether block copolymers and polyether segments are exemplified by the block copolymers having the formulas: 
in which x, y, z, i, and j have values from about 2 to about 800, preferably from about 5 to about 200, more preferably from about 5 to about 80, and wherein for each R1, R2 pair, one is hydrogen and the other is a methyl group. Formulas (XXIV) through (XXVI) are oversimplified in that, in practice, the orientation of the isopropylene radicals within the B block will be random. This random orientation is indicated in formulas (XXVII) and (XXVIII), which are more complete. Such poly(oxyethylene)-poly(oxypropylene) block copolymers have been described by Santon, Am. Perfumer Cosmet., 72(4):54-58 (1958); Schmolka, Loc. cit. 82(7):25-30 (1967); Non-ionic Surfactants, Schick, ed. (Dekker, N.Y., 1967), pp. 300-371. A number of such compounds are commercially available under such generic trade names as xe2x80x9clipoloxamersxe2x80x9d, xe2x80x9cpoloxamersxe2x80x9d, xe2x80x9cPluronic(copyright)xe2x80x9d, and xe2x80x9csynperonics.xe2x80x9d poly(oxyethylene)-poly(oxypropylene) polymers within the B-A-B formula are often referred to as xe2x80x9creversedxe2x80x9d Pluronic(copyright), xe2x80x9cPluronic-R(copyright)xe2x80x9d or xe2x80x9cmeroxapol.xe2x80x9d
The xe2x80x9cpolyoxaminexe2x80x9d polymer of formula (XXVII) is available from BASF (Wyandotte, Mich.) under the tradename Tetronic(copyright). The order of the polyoxyethylene and polyoxypropylene blocks represented in formula (XXVII) can be reversed, creating Tetronic-R(copyright) of formula (XXVIII) also available from BASF. See, Schmolka, J. Am. Oil. Soc., 59:110 (1979). Polyoxypropylene-polyoxyethylene block copolymers can also be designed with hydrophilic blocks comprising a random mix of ethylene oxide and propylene oxide repeating units. To maintain the hydrophilic character of the block, ethylene oxide will predominate. Similarly, the hydrophobic block can be a mixture of ethylene oxide and propylene oxide repeating units. Such block copolymers are available from BASF under the tradename Pluradot(trademark).
A number of pluronics are designed to meet the following formula: 
Of course, those skilled in the art will recognize that the values of m and n will usually represent a statistical average and that the number of repeating units of the first block of a given molecule will generally not be exactly the number of repeating units of the third block. The characteristics of a number of block copolymers, described with reference to formula (XXIX), are as follows:
Some other specific poly(oxyethylene)-poly(oxypropylene) block copolymers relevant to the invention include:
The diamine-linked block copolymer of formula (XXVII) can also be a member of the family of diamine-linked polyoxyethylene-polyoxypropylene polymers of formula: 
wherein the dashed lines represent symmetrical copies of the polyether extending off the second nitrogen, R* an alkylene of about 2 to about 6 carbons, a cycloalkylene of about 5 to about 8 carbons or phenylene, for R1 and R2, either (a) both are hydrogen or (b) one is hydrogen and the other is methyl, for R3 and R4 either (a) both are hydrogen or (b) one is hydrogen and the other is methyl, if both of R3 and R4 are hydrogen, then one R5 and R6 is hydrogen and the other is methyl, and if one of R3 and R4 is methyl, then both of R5 and R6 are hydrogen.
The hydrophobic/hydrophilic properties of a given block copolymer depends upon the ratio of the number of oxypropylene groups to the number of oxypropylene groups. For a composition containing a single block copolymer of poly(oxyethylene)-poly(oxypropylene), for example, this relationship, taking into account the molecular masses of the central hydrophobic block and the terminal hydrophilic blocks, can be expressed as follows:   n  =            H      L        ·    1.32  
in which H is the number of oxypropylene units and L is the number of oxyethylene units. In the general case of a block copolymer containing hydrophobic B-type segments and hydrophilic A-type segments, the hydrophobic-hydrophilic properties and micelle-forming properties are related to the value n as defined as:
n=(|B|/|A|)xc3x97(b/a)
where |B| and |A| are the number of repeating units in the hydrophobic and hydrophilic blocks of the copolymer, respectively, and b and a are the molecular weights for the respective repeating units.
Selecting a block copolymer with the appropriate n value will depend upon the hydrophobic/hydrophilic properties of the specific agent, or the composite hydrophilic/hydrophilic properties of a mixture of agents to be formulated. Typically, n will range in value from about 0.2 to about 9.0, more preferably between about 0.25 and about 1.5. This range should be viewed not as numerically critical but as expressing the optimum hydrophobic/hydrophilic balance between the predominantly hydrophilic poly(oxyethylene) blocks, and the predominantly hydrophobic poly(oxypropylene) blocks.
An important aspect of the present invention-involves utilizing mixture of different block-copolymers of poly(oxyethylene)-poly(oxypropylene) to achieve a more specific hydrophobic-hydrophilic balance suitable for a given cytokine or mixture of several cytokines, preserving the optimal size of particles. For example, a first block copolymer may have an n of 1.0 whereas a second may have a value of 1.5. If material having an n of 1.3 is desired, a mixture of one weight portion of the first block copolymer and 1.5 weight portion of the second block-copolymer can be employed.
Thus, a more generalized relationship for such mixtures can be expressed as follows:   N  =      1.32    ·          [                                                  H              1                        ·                          m              1                                                          (                              L                1                            )                        ·                          (                                                m                  1                                +                                  m                  2                                            )                                      +                                            H              2                        ·                          m              2                                                          (                              L                2                            )                        ·                          (                                                m                  1                                +                                  m                  2                                            )                                          ]      
in which H1 and H2 are the number of oxypropylene units in the first and second block copolymers, respectively; L1 is the number of oxyethylene units in the first block copolymer; L2 is the number of oxyethylene units in the second block copolymer; m1 is the weight proportion in the first block-copolymer; and m2 is the weight proportion in the second block copolymer.
An even more general case of a mixture of K block copolymers containing hydrophobic B-type block copolymers and hydrophilic A-type block copolymers, the N value can be expressed as follows:   N  =            b      a        ⁢                  ∑                  i          =          1                k            ⁢              xe2x80x83            ⁢              (                                                            "LeftBracketingBar"                B                "RightBracketingBar"                            i                                                      "LeftBracketingBar"                A                "RightBracketingBar"                            i                                ,                                    m              i                        M                          )            
where |A|i and |B|i are the numbers of repeating units in the hydrophilic (A-type) and hydrophobic (B-type) blocks of the i-th block copolymer, m is the weight proportion of this block copolymers, M is the sum of weight proportions of all block copolymers in the mixture       (          M      =                        ∑                      i            =            1                    k                ⁢                  xe2x80x83                ⁢                  m          i                      )    ,
and a and b are the molecular weights for the repeating units of the hydrophilic and hydrophobic blocks of these block copolymers respectively.
If only one block copolymer of poly(oxyethylene)-poly(oxypropylene) is utilized, N will equal n. An analogous relationship will apply to compositions employing more than two block copolymers of poly(oxyethylene)-poly(oxypropylene).
Where mixtures of block copolymers are used, a value N will be used, which value will be the weighted average of n for each contributing copolymers, with the averaging based on the weight portions of the component copolymers. The value N can be used to estimate the micelle-forming properties of a mixture of copolymers. The use of the mixtures of block copolymers enhances solubility and prevents aggregation of more hydrophobic block copolymers in the presence of the serum proteins. Particularly, poly(oxyethylene)-poly(oxypropylene) block copolymers with the ethylene oxide content of more than 50% solubilize hydrophobic block copolymers with ethylene oxide content of no more than 50%. In such mixtures, the preferred ratio of the hydrophilic and hydrophobic copolymer is at least 2:1 (w/w), preferably at least 5:1 (w/w), still more preferably at least 8:1 (w/w).xe2x80x9d When copolymers other than polyethylene oxide-polypropylene oxide copolymers are used, similar approaches can be developed to relate the hydrophobic/hydrophilic properties of one member of the class of polymers to the properties of another member of the class.
Using the above parameters, one or more block copolymers of poly(oxyethylene)-poly(oxypropylene) are combined so as to have a value for N of from about 0.1 to about 9, more preferably from about 0.25 to about 1.5. The combined copolymers form micelles, the value of N affecting in part the size of the micelles thus produced. Typically, the micelles will have an average diameter of from about 10 to about 25 nm, although this range can vary widely. The average diameter of any given preparation can be readily determined by quasi-elastic light scattering techniques.
In another aspect, the invention relates to a polynucleotide complex comprising a block copolymer at least one of formulas (I)-(XIII), wherein the A-type and B-type blocks are substantially made up of repeating units of formula xe2x80x94Oxe2x80x94R9, where R9 is:
(1) xe2x80x94(CH2)nxe2x80x94CH(R6), wherein n is an integer from 0 to about 5 and R6 is hydrogen, cycloalkyl having 3-8 carbon atoms, alkyl having 1-6 carbon atoms, phenyl, alkylphenyl wherein the alkyl has 1-6 carbon atoms, hydroxy, hydroxyalkyl, wherein the alkyl has 1-6 carbon atoms, alkoxy having 1-6 carbon atoms, an alkyl carbonyl group having 2-7 carbon atoms, alkoxycarbonyl, wherein the alkoxy has 1-6 carbon atoms, alkoxycarbonylalkyl, wherein the alkoxy and alkyl each independently has 1-6 carbon atoms, alkylcarboxyalkyl, wherein each alkyl group has 1-6 carbon atoms, aminoalkyl wherein the alkyl group has 1-6 carbon atoms, alkylamine or dialkylamino, wherein each alkyl independently has 1-6 carbon atoms, mono- or di-alkylaminoalkyl wherein each alkyl independently has 1-6 carbon atoms, chloro, or chloroalkyl wherein the alkyl has from 1-6 carbon atoms, fluoro, fluoroalkyl wherein the alkyl has from 1-6 carbon atoms, cyano or cyano alkyl wherein the alkyl has from 1-6 carbon atoms or carboxyl; (2) a carbocyclic group having 3-8 ring carbon atoms, wherein the group can be for example, cycloalkyl or aromatic groups, and which can include alkyl having 1-6 carbon atoms, alkoxy having 1-6 carbon atoms, alkylamino having 1-6 carbon atoms, dialkylamino wherein each alkyl independently has 1-6 carbon atoms, amino, sulfonyl, hydroxy, carboxy, fluoro or chloro substitutions, or (3) a heterocyclic group, having 3-8 ring atoms, which can include heterocycloalkyl or heteroaromatic groups, which can include from 1-4 heteroatoms selected from the group consisting of oxygen, nitrogen, sulfur, and mixtures thereof, and which can include alkyl of 1-6 carbon atoms, alkoxy having 1-6 carbon atoms, alkylamino having 1-6 carbon atoms, dialkylamino wherein each alkyl independently has 1-6 carbon atoms, amino, sulfonyl, hydroxy, carboxy, fluoro, or chloro substitutions.
Preferably, n is an integer from 1 to 3. The carbocyclic or heterocyclic groups comprising R5 preferably have from 4-7 ring atoms, more preferably 5-6. Heterocycles preferably include from 1-2 hetero atoms, more preferably, the heterocycles have one heteroatom. Preferably, the heterocycle is a carbohydrate or carbohydrate analog. Those of ordinary skill will recognize that the monomers required to make these polymers are synthetically available. In some cases, polymerization of the monomers will require the use of suitable protective groups, as will be recognized by those of ordinary skill in the art. Generally, the A- and B-type blocks are at least about 80% comprised of xe2x80x94OR5xe2x80x94 repeating units, more preferably at least about 90%, yet more preferably at least about 95%.
In another aspect, the invention relates to a polynucleotide complex comprising a block copolymer of one of formulas (I)-(XIII) wherein the A-type and B-type blocks consist essentially of repeating units of formula xe2x80x94Oxe2x80x94R5 wherein R7 is a C to C alkyl group.
The block copolymers utilized in the invention will typically, under certain circumstances, form micelles of from about 10 nm to about 100 nm in diameter. Micelles are supramolecular complexes of certain amphiphilic molecules that form in aqueous solutions due to microphase separation of the nonpolar portions of the amphiphiles. Micelles form when the concentration of the amphiphile reaches, for a given temperature, a critical micellar concentration (xe2x80x9cCMCxe2x80x9d) that is characteristic of the amphiphile. Such micelles will generally include from about 10 to about 300 block copolymers. By varying the sizes of the hydrophilic and hydrophobic portions of the block copolymers, the tendency of the copolymers to form micelles at physiological conditions can be varied. The micelles have a dense core formed by the water insoluble repeating units of the B blocks and charge-neutralized nucleic acids, and a hydrophilic shell formed by the A blocks. The micelles have translational and rotational freedom in solution, and solutions containing the micelles have low viscosity similar to water. Micelle formation typically occurs at copolymer concentrations from about 0.001 to 5% (w/v). Generally, the concentration of polycationic polymers and polynucleic acid will be less than the concentration of copolymers in the polynucleotide compositions, preferably at least about 10-fold less, more preferably at least about 50-fold.
At high concentrations, some of the block copolymers utilized in the invention will form gels. These gels are viscous systems in which the translational and rotational freedom of the copolymer molecules is significantly constrained by a continuous network of interactions among copolymer molecules. In gels, microsegregation of the B block repeating units may or may not occur. To avoid the formation of gels, polymer concentrations (for both block copolymers and polyether/polycation polymers) will preferably be below about 15% (w/v), more preferably below about 10%, still more preferably below about 5%. In the first embodiment of the invention, it is more preferred that gels be avoided.
When the polynucleotide composition includes cationic components, the cations will associate with the phosphate groups of the polynucleotide, neutralizing the charge on the phosphate groups and rendering the polynucleotide component more hydrophobic. The neutralization is preferably supplied by cations on R-type polymeric segments or on polycationic polymers. However, the phosphate charge can also be neutralized by chemical modification or by association with a hydrophobic cations such as N-[1-(2,3-dioleyloxy)-N,Nxe2x80x2-3-methylammonium chloride]. In aqueous solution, the charge neutralized polynucleotides are believed to participate in the formation of supramolecular, micelle-like particles, termed xe2x80x9cpolynucleotide complexes.xe2x80x9d The hydrophobic core or the complex comprises the charge neutralized polynucleotides and the B-type copolymer blocks. The hydrophilic shell comprises the A-type copolymer blocks. The size of the complex will generally vary from about 10 nm to about 100 nm in diameter. In some contexts, it is practical to isolate the complex from unincorporated components. This can be done, for instance, by gel filtration chromatography.
The ratio of the components of the polynucleotide composition is an important factor in optimizing the effective transmembrane permeability of the polynucleotides in the composition. This ratio can be identified as ratio Ø, which is the ratio of positively charged groups to negatively charged groups in the composition at physiological pH. If Ø less than 1, the complex contains non-neutralized phosphate from the polynucleotide. The portions of the polynucleotides adjacent to the non-neutralized charges are believed to be a part of the shell of a polynucleotide complex. Correspondingly, if Ø greater than 1, the polycationic polymer or R-type segment will have non-neutralized charges, and the un-neutralized portions will fold so that they form a part of the shell of the complex. Generally, Ø will vary from about 0 (where there are no cationic groups) to about 100, preferably Ø will range between about 0.01 and about 50, more preferably, between about 0.1 and about 20. Ø can be varied to increase the efficiency of transmembrane transport and, when the composition comprises polynucleotide complexes, to increase the stability of the complex. Variations in Ø can also affect the biodistribution of the complex after administration to an animal. The optimal Ø will depend on, among other things, (1) the context in which the polynucleotide composition is being used, (2) the specific polymers and oligonucleotides being used, (3) the cells or tissues targeted, and (4) the mode of administration.
Surfactant-Containing Polynucleotide Compositions. The invention also includes compositions of polynucleotides, cationic copolymer, and a suitable surfactant. The surfactant, should be (i) cationic (including those used in various transfection cocktails), (ii) nonionic (e.g., Pluronic or Tetronic), or (iii) zwitterionic (including betains and phospholipids). These surfactants increase solubility of the complex and increase biological activity of the compositions.
Suitable cationic surfactants include primary amines, secondary amines, tertiary amines (e.g., N,Nxe2x80x2,Nxe2x80x2-polyoxyethylene(10)-N-tallow-1,3-diaminopropane), quaternary amine salts (e.g., dodecyltrimethylammonium bromide, hexadecyltrimethylammonium bromide, mixed alkyltrimethylammonium bromide, tetradecyltrmethylammonium bromide, benzalkonium chloride, benzethonium chloride, benzyldimethyldodecylammonium chloride, benzyldimethylhexadecylammonium chloride, benzyltrimethylammonium methoxide, cetyldimethylethylammonium bromide, dimethyldioctadecyl ammonium bromide, methylbenzethonium chloride, decamethonium chloride, methyl mixed trialkyl ammonium chloride, methyl trioctylammonium chloride), N,N-dimethyl-N-[2-(2-methyl-4-(1,1,3,3-tetramethylbutyl)-phenoxy]ethoxy)ethyl]-benzenemeth-anaminium chloride (DEBDA), dialkyldimetylammonium salts, N-[1-(2,3-dioleyloxy)-propyl]-N,N,N,-trimethylammonium chloride, 1,2-diacyl-3-(trimethylammonio)propane (acyl group=dimyristoyl, dipalmitoyl, distearoyl, dioleoyl), 1,2-diacyl-3-(dimethylammonio)propane (acyl group=dimyristoyl, dipalmitoyl, distearoyl, dioleoyl), 1,2-dioleoyl-3-(4xe2x80x2-trimethylammonio) butanoyl-sn-glycerol, 1,2-dioleoyl-3-succinyl-sn-glycerol choline ester, cholesteryl (4xe2x80x2-trimethylammonio) butanoate), N-alkyl pyridinium salts (e.g. cetylpyridinium bromide and cetylpyridinium chloride), N-alkylpiperidinium salts, dicationic bolaform electrolytes (C12Me6; C12Bu6), dialkylglycetylphosphorylcholine, lysolecithin, L-xcex1-dioleoyl phosphatidylethanolamine), cholesterol hemisuccinate choline ester, lipopolyamines (e.g., dioctadecylamidoglycylspermine (DOGS), dipalmitoyl phosphatidylethanolamidospermine (DPPES), lipopoly-L(or D)-lysine (LPLL, LPDL), poly(L (or D)-lysine conjugated to N-glutarylphosphatidylethanolamine, didodecyl glutamate ester with pendant amino group (C12GluPhCnN+), ditetradecyl glutamate ester with pendant amino group (C14GluCnN+), cationic derivatives of cholesterol (e.g., cholesteryl-3xcex2-oxysuccinamidoethylenetrimethylammonium salt, cholesteryl-3xcex2-oxysuccinamidoethylenedimethylamine, cholesteryl-3xcex2-carboxyamidoethylenetrimethylammonium salt, cholesteryl-3xcex2-carboxyamidoethylenedimethylamine, 3xcex2[N-(Nxe2x80x2,Nxe2x80x2-dimethylaminoetane-carbomoil]cholesterol).
Suitable non-ionic surfactants include n-Alkylphenyl polyoxyethylene ether, n-alkyl polyoxyethylene ethers (e.g., Tritons(trademark)), sorbitan esters (e.g., Spans(trademark)), polyglycol ether surfactants (Tergitol(trademark)), polyoxyethylenesorbitan (e.g., Tweens(trademark)), polysorbates, polyoxyethylated glycol monoethers (e.g., Brij(trademark), polyoxylethylene 9 lauryl ether, polyoxylethylene 10 ether, polyoxylethylene 10 tridecyl ether), lubrol, copolymers of ethylene oxide and propylene oxide (e.g., Pluronic(trademark), Pluronic R(trademark), Teronic(trademark), Pluradot(trademark)), alkyl aryl polyether alcohol (Tyloxapol(trademark)), perfluoroalkyl polyoxylated amides, N,N-bis[3-D-gluconamidopropyl]cholamide, decanoyl-N-methylglucamide, n-decyl xcex1-D-glucopyranozide, n-decyl xcex2-D-glucopyranozide, n-decyl xcex2-D-maltopyranozide, n-dodecyl xcex2-D-glucopyranozide, n-undecyl xcex2-D-glucopyranozide, n-heptyl xcex2-D-glucopyranozide, n-heptyl xcex2-D-thioglucopyranozide, n-hexyl xcex2-D-glucopyranozide, n-nonanoyl xcex2-D-glucopyranozide 1-monooleyl-rac-glycerol, nonanoyl-N-methylglucamide, n-dodecyl xcex1-D-maltoside, n-dodecyl xcex2-D-maltoside, N,N-bis[3-gluconamidepropyl]deoxycholamide, diethylene glycol monopentyl ether, digitonin, heptanoyl-N-methylglucamide, heptanoyl-N-methylglucamide, octanoyl-N-methylglucamide, n-octyl xcex2-D-glucopyranozide, n-octyl xcex1-D-glucopyranozide, n-octyl xcex2-D-thiogalactopyranozide, n-octyl xcex2-D-thioglucopyranozide.
Suitable Zwitterionic surfactants include betaine (R1R2R3N+Rxe2x80x2CO2xe2x88x92, where R1R2R3Rxe2x80x2 are hydrocarbon chains and R1 is the longest one), sulfobetaine (R1R2R3N+Rxe2x80x2SO3xe2x88x92), phospholipids (e.g., dialkyl phosphatidylcholine), 3-[(3-cholamidopropyl)-dimethylammonio]-2-hydroxy-1-propanesulfonate, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate, N-decyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, N-octadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, N-octyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, N-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, and dialkyl phosphatitidylethanolamine.
Nucleic acids. A wide variety of nucleic acid molecules can be the nucleic acid component of the compositions. These include natural and synthetic DNA or RNA molecules and nucleic acid molecules that have been covalently modified (to incorporate groups including lipophilic groups, photo-induced crosslinking, groups, alkylating groups, organometallic groups, intercalating groups, lipophilic groups, biotin, fluorescent, and radioactive groups, and groups that modify the phosphate backbone). Such nucleic acid molecules can be, among other things, antisense nucleic acid molecules, gene-encoding DNA (usually including an appropriate promoter sequence), ribozymes, aptamers, anitgen nucleic acids, oligonucleotide xcex1-anomers, ethylphosphotriester analogs, alkylphosphomates, phosphorothionate and phosphorodithionate oligonucleotides, and the like. In fact, the nucleic acid component can be any nucleic acid that can beneficially be transported into a cell with greater efficiency, or stabilized from degradative processes, or improved in its biodistribution after administration to an animal.
Targeting molecules. It will in some circumstances be desirable to incorporate, by noncovalent association, targeting molecules. See for example, Kabanov et al., J. Controlled Release, 22:141 (1992), the contents of which are hereby incorporated by reference. The targeting molecules that can be associated with the composition typically have a targeting group having affinity for a cellular site and a hydrophobic group. The targeting molecule will spontaneously associate with the polynucleotide complex and be xe2x80x9canchoredxe2x80x9d thereto through the hydrophobic group. These targeting adducts will typically comprise about 10% or less of the copolymers in a composition.
In the targeting molecule, the hydrophobic group can be, among other things, a lipid group such as a fatty acyl group. Alternately, it can be a block copolymer or another natural synthetic polymer. The targeting group of the targeting molecule will frequently comprise an antibody, typically with specificity for a certain cell surface antigen. It can also be, for instance, a hormone having a specific interaction with a cell surface receptor, or a drug having a cell surface receptor. For example, glycolipids could serve to target a polysaccharide receptor. It should be noted that the targeting molecule can be attached to any of the polymer blocks identified herein, including R-type polymeric blocks and to the polycationic polymers. For instance, the targeting molecule can be covalently attached to the free-terminal groups of the polyether segment of the block copolymer of the invention. Such targeting molecules can be covalently attached to the xe2x80x94OH end group of the polymers of the formulas XVIII, XIX, XX, and XXI, and the xe2x80x94NH2 end group of the polymers of formulas XVIII (preferably the xcex5-amino group of the terminal lysyl residue), XX or XXIII, or the xe2x80x94COOH end group of the polymers of formulas XVIII and XIX. Targeting molecules can be used to facilitate intracellular transport of the polynucleotide composition, for instance transport to the nucleus, by using, for example, fusogenic peptides as targeting molecules described by Soukchareun et al., Bioconjugate Chem., 6:43 (1995), or Arar et al., Bioconjugate Chem., 6:43 (1995), caryotypic peptides, or other biospecific groups providing site-directed transport into a cell (in particular, exit from endosomic compartments into cytoplasm, or delivery to the nucleus).
The polynucleotide component of the compositions can be any polynucleotide, but are preferably a polynucleotide with at least about 3 bases, more preferably at least about 5 bases. Still more preferred are at least 10 bases. Included among the suitable polynucleotides are viral genomes and viruses (including the lipid or protein viral coat). This includes viral vectors including, but not limited to, retroviruses, adenoviruses, herpes-virus, or Pox-virus. Other suitable viral vectors for use with the present invention will be obvious to those skilled in the art. The terms xe2x80x9cpoly(nucleic acid)xe2x80x9d and xe2x80x9cpolynucleotidexe2x80x9d are used interchangeably herein. An oligonucleotide is a polynucleotide, as are DNA and RNA.
A polynucleotide derivative is a polynucleotide having one or more moieties (i) wherein the moieties are cleaved, inactivated or otherwise transformed so that the resulting material can function as a polynucleotide, or (ii) wherein the moiety does not prevent the derivative from functioning as a polynucleotide.
Therapeutic applications. The present compositions can be used in a variety of treatments. For example, the compositions can be used in gene therapy including gene replacement or excision therapy, and gene addition therapy (B. Huber, Gene therapy for neoplastic diseases; B. E. Huber and J. S. Lazo Eds., The New York Academy of Sciences, N.Y., N.Y., 1994, pp. 6-11). Also, antisense therapy targets genes in the nucleus and/or cytoplasm of the cell, resulting in their inhibition (Stein and Cheng, Science 261:1004 (1993); De Mesmaeker et al., Acc. Chem. Res., 28:366 (1995)). Aptamer nucleic acid drugs target both intra-and extracellular proteins, peptides and small molecules. See Ellington and Szostak, Nature (London), 346:818 (1990). Antigen nucleic acid compounds can be used to target duplex DNA in the nucleus. See Helene and Tolume, Biochim, Biophys., Acta 1049:99 (1990). Catalytic polynucleotides target mRNA in the nucleus and/or cytoplasm. Cech, Curr. Opp. Struct. Biol., 2:605 (1992).
Examples of genes to be replaced, inhibited and/or added include, adenosine deaminase, tumor necrosis factor, cell growth factors, Factor IX, interferons (such as xcex1-, xcex2-, and xcex3-interferon), interleukins (such interleukin 2, 4, 6, and 12), HLA-B7, HSV-TK, CFTR, HIV-1, xcex2-2, microglobulin, retroviral genes (such as gag, pol, env, tax, and rex), cytomegalovirus, herpes viral genes (such as herpes simplex virus type I and II genes ICP27/UL54, ICP22/US1, ICP/IE175, protein kinase and exonuclease/UL13, protein kinase/US3, ribonuclease reductase ICP6/UL39, immediate early (IE) mRNA IE3/IE175/ICP4, 1E4/ICP22/US1, IE5/ICP47, IE110, DNA polymerase/UL30, UL13), human multidrug resistance genes (such as mdrl), oncogenes (such as H-c-ras, c-myb, c-myb, bcl-2, bcr/abl), tumor suppressor gene p53, human MHC genes (such as class 1 MHC), immunoglobulins (such as IgG, IgM, IgE, IgA), hemoglobin xcex1- and xcex2-chains, enzymes (such as carbonic anhydrase, triosephoshate isomerase, GTP-cyclhydrdolase I, phenylalanine hydrolase, sarcosine dehydrogenase, glucocerobrosidase, glucose-6-phosphste dehydrogenase), dysotrophin, fibronectin, apoliprotein E, cystic fibrosis transmembrane conductance protein, c-src protein, V(D)J recombination activating protein, immunogenes, peptide and protein antigens (xe2x80x9cDNA vaccinesxe2x80x9d) and the like.
Genetic diseases can also be treated by the instant compositions. Such diseases include, rheumatoid arthritis, psoriasis, Crohn""s disease, ulcerative colitis, xcex1-thalassemia, xcex2-thalassemia, carbonic anhydrase II deficiency syndrome, triosephosphate isomerase deficiency syndrome, tetrahydrobiopterindeficient hyperphenylalaniemia, classical phenylketonuria, muscular dystrophy such as Duchenne Muscular Dystrophy, hypersarkosinemia, adenomatous intestinal polyposis, adenosine deaminase deficiency, malignant melanoma, glucose-6-phosphste dehydrogenase deficiency syndrome, arteriosclerosis and hypercholesterolemia, Gaucher""s disease, cystic fibrosis, osteopetrosis, increased spontaneous tumors, T and B cell immunodeficiency, high cholesterol, arthritis including chronic rheumatoid arthritis, glaucoma, alcoholism and the like.
The compositions can also be used to treat neoplastic diseases including, but not limited to, breast cancer (e.g., breast, pancreatic, gastric, prostate, colorectal, lung, ovarian), lymphomas (such as Hodgkin and non-Hodgkin lymphoma), melanoma and malignant melanoma, advanced cancer hemophilia B, renal cell carcinoma, gliblastoma, astrocytoma, gliomas, AML and CML and the like.
Additionally, the compositions can be used to treat (i) cardiovascular diseases including but not limited to stroke, cardiomyopathy associated with Duchenne Muscular Dystrophy, myocardial ischemia, restenosis and the like, (ii) infectious diseases such as Hepatitis, HIV infections and AIDS, Herpes, CMV and associated diseases such as CMV renitis, (iii) transplantation related disorders such as renal transplant rejection and the like, and (iv) are useful in vaccine therapies and immunization, including but not limited to melanoma vaccines, HIV vaccines, malaria, tuberculosis, and the like.
Target Cells. Cell targets can be ex vivo and/or in vivo, and include T and B lymphocytes, primary CML, tumor infiltrating lymphocytes, tumor cells, leukemic cells (such as HL-60, ML-3, KG-1 and the like), skin fibroblasts, myoblasts, cells of central nervous system including primary neurons, liver cells, carcinoma (such as Bladder carcinoma T24, human colorectal carcinoma Caco-2), melanoma, CD34+ lymphocytes, NK cells, macrophages, hemotopoetic cells, neuroblastona (such as LAN-5 and the like), gliomas, lymphomas (such as Burkitt lymphomas ST486), JD38), T-cell hybridomas, muscle cells such as primary smooth muscle, and the like.
Methods of use. The polynucleotide compositions of the invention can be administered orally, topically, rectally, vaginally, by pulmonary route by use of an aerosol, or parenterally, i.e. intramuscularly, subcutaneously, intraperitoneallly or intravenously. The polynucleotide compositions can be administered alone, or it can be combined with a pharmaceutically-acceptable carrier or excipient according to standard pharmaceutical practice. For oral administration, the polynucleotide compositions can be used in the form of tablets, capsules, lozenges, troches, powders, syrups, elixirs, aqueous solutions and suspensions, and the like. In the case of tablets, carriers that can be used include lactose, sodium citrate and salts of phosphoric acid. Various disintegrants such as starch, and lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc, are commonly used in tablets. For oral administration in capsule form, useful diluents are lactose and high molecular weight polyethylene glycols. When aqueous suspensions are required for oral use, the polynucleotide compositions can be combined with emulsifying and suspending agents. If desired, sweetening and/or flavoring agents can be added. For parenteral administration, sterile solutions of the conjugate are usually prepared, and the pH of the solutions are suitably adjusted and buffered. For intravenous use, the total concentration of solutes should be controlled to render the preparation isotonic. For ocular administration, ointments or droppable liquids may be delivered by ocular delivery systems known to the art such as applicators or eye droppers. Such compositions can include mucomimetics such as hyaluronic acid, chondroitin sulfate, hydroxypropyl methylcellulose or poly(vinyl alcohol), preservatives such as sorbic acid, EDTA or benzylchronium chloride, and the usual quantities of diluents and/or carriers. For pulmonary administration, diluents and/or carriers will be selected to be appropriate to allow the formation of an aerosol.
For intramuscular administration, the formulation of the polynucleotides will be without any polycationic moiety since naked polynucleotides itself can be transferred and expressed in muscle without any polycation-containing delivery systems. The muscle has the following features: unique cytoarchitecture, multiple nuclei per myotubes, specific-polynucleotides binding proteins (triadin), and unique nucleocytoplasmic transport. At present, it is still unclear as to which features listed above may be responsible for the uptake and expression of naked polynucleotides in muscle. Cationic complexes of polynucleotides have been shown to enhance uptake and gene expression in virtually all tissue types but surprisingly the same complexes do not contribute to a better uptake and gene expression in muscle. In fact, cationic complexation of polynucleotides inhibit uptake and gene expression in muscle and reported by several laboratories. Thus, for intramuscular injection of polynucleotides, complexation of polynucleotides should be avoided. This invention uses nonionic block copolymers for intramuscular delivery of polynucleotides. Block copolymers alone are totally inefficient at transferring genetic material in cells in vitro and in vivo (see example 42). Moreover, unlike polycation-containing block copolymers, the above nonionic block copolymers do not increase gene expression in the peripheral organs such as lungs, liver, kidneys.
The following examples will serve to further typify the nature of the invention but should not be construed as a limitation on the scope thereof, which is defined solely by the appended claims.